Cross-validated methods for promoter/transcription start site mapping in SL trans-spliced genes, established using the Ciona intestinalis troponin I gene

• Published in: Nucleic acids research Vol. 39; no. 7; pp. 2638 - 2648
• Main Authors: Khare, Parul, Mortimer, Sandra I, Cleto, Cynthia L, Okamura, Kohji, Suzuki, Yutaka, Kusakabe, Takehiro, Nakai, Kenta, Meedel, Thomas H, Hastings, Kenneth E M
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.2011
• Subjects: 5' Untranslated Regions; Animals; Chordata; Chromosome Mapping - methods; Ciona intestinalis; Ciona intestinalis - genetics; Genomics; High-Throughput Nucleotide Sequencing; Marine; Promoter Regions, Genetic; Sequence Analysis, RNA; TATA Box; Trans-Splicing; Transcription Initiation Site; Troponin I - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkq1151

In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5'-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5'-leader trans-splicing (SL trans-splicing) because the original 5'-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5'-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA's original 5'-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla.